ES2319475B1 - BIOACTIVE PEPTIDES IDENTIFIED IN ENZYMATIC HYDROLYZES OF LACTEE CASEINS AND PROCEDURE OF OBTAINING. - Google Patents

Abstract

Bioactive peptides identified in Enzymatic hydrolysates of milk caseins and the process of obtaining.

The invention consists in the production of bioactive products derived from milk proteins for the production of bioactive lactoproducts derived from milk proteins, especially caseins. The ten peptides object of the patent can be obtained chemically, biotechnologically or by enzymatic treatment from proteins that contain them and give rise to peptides with antimicrobial activity, inhibitor of the anqiotensin converting enzyme in vitro and / or antihypertensive activity and / or antioxidant activity. These nutraceutical products, either as hydrolyzate or as biactive peptides, are both useful for the food industry and for the pharmaceutical industry.

Description

Bioactive peptides identified in Enzymatic hydrolysates of milk caseins and the process of obtaining.

Technical sector

The invention consists in the production of bioactive products derived from milk proteins. These proteins give rise, after an enzymatic treatment, to peptides with antimicrobial activity and / or angiotensin converting enzyme (ACEI activity) inhibitor activity in vitro and / or antihypertensive activity and / or antioxidant activity, which can be applied to the food industry and pharmaceutical.

State of the art

The role of milk in human nutrition is essential from the moment of birth and constitutes a food With a high nutritional and functional value. The recent development of new biotechnological and separation techniques allow the fractionation of different milk components to be used for new food or non-food purposes and, of in this way, new applications that contribute to Increase your consumption. Thus, different companies dedicated to the production of isolated proteins from fractions of the milk, are interested in increasing and diversifying the uses of some components, such as caseins and seroproteins. This is the case of the industries dedicated to the production of lactoferrin, used as an antimicrobial agent and already used in baby foods, yogurts, nutritional supplements, special formulations, dental and dermatological products. The Lactoferrin is also used for its antimicrobial activity as Additive in fresh milk to extend its duration.

In recent years, functional foods have broken into the food sector, due to the increased awareness of consumers of the relationship between diet and health. Within the functional ingredients, defined as those components that, incorporated into the food, exercise specific biological activities that go beyond mere nutritional role, occupy a prominent place, due to their diversity and multifunctionality, bioactive peptides. These peptides correspond to inactive fragments within the precursor protein, but which, after being released by hydrolysis processes in vivo and / or in vitro , exert different physiological functions in the body. Since its discovery, in 1979, peptides derived from food proteins with different biological activities have been described: antimicrobial, antihypertensive, immunomodulant, antithrombotic, opioid, antioxidant, etc. These peptides have a potential food and pharmaceutical use and can be released by various strategies, with enzymatic hydrolysis and microbial fermentation being the most used in the
present.

Among the bioactive peptides, those that exert antimicrobial properties (R. Floris, I. Recio, B. Berkhout, and S. Visser, Antibacterial and antiviral effects of milk proteins and derivatives thereof, Current Pharmaceutical Design, 2003, 9: 1257 -1275). The antimicrobial activity of milk has been studied for a long time and has traditionally been attributed to different proteins with antimicrobial activity present in this food (immunoglobulins, lactoferrin, lactoperoxidase, lysozyme, etc.). However, recently, the antimicrobial activity of peptides derived from milk proteins has also been demonstrated. Although so far there are no conclusive studies on the mechanism of action of antimicrobial peptides derived from milk proteins, preliminary results have described the ability of some of these bioactive sequences to interact and lyse bacterial membranes (D. Chapple, DJ Mason, CL Joannou, EW Odell, V. Gant, RW Evans, Structure-function relationship of antibacterial synthetic peptides homologous to a helical surface region on human lactoferrin against Escherichia coli serotype O111, Infection and Immunity, 1998, 66, 2434-2440). Peptides with antimicrobial activity obtained from enzymatic hydrolysates of caseins of bovine origin have been described, such as? S2 -casein (EP 1114060, Process for producing cationic peptides from biological fluids) and? -Casein and? -casein (WO99 / 26971, Antimicrobial peptides). By similar hydrolysis processes, peptides with antimicrobial properties derived from whey proteins, such as lactoferrin (WO2004 / 089986, Antimicrobial peptide from transferrin family) have been isolated and identified.

Another group of bioactive peptides of great importance is that of peptides with antihypertensive activity given the high incidence of coronary diseases related to hypertension in developed countries. Many of these peptides act by regulating the renin-angiotensin system by inhibiting the angiotensin-converting enzyme (RCT) (T. Takano, Milk derived peptides and hypertension reduction, International Dairy Journal, 1998, 8: 375-381) although not rule out that they can act through other mechanisms. Different peptides with ACE inhibitory activity (ACEI), obtained from enzymatic casein hydrolysates (US 6514941, Method of preparing a casein hydrolysate enriched in anti-hypertensive peptides) and whey proteins (WO01 / 85984) have been discovered , Enzymatic treatment of whey proteins for the production of antihypertensive peptides, the resulting products and treatment of hypertension in mammals). Studies on the structure / activity relationship of peptides with antihypertensive activity have revealed the fundamental role of certain hydrophobic amino acids in the development of this activity (H.-S. Cheung, F.-L. Wang, MA Ondetti, EF Sabo , and DW Cushman, Binding of peptide substrates and inhibitors of angiotensin-converting enzyme. Importance of the COOH-terminal dipeptide sequence. Journal of Biological Chemistry 1980, 255: 401-407). The presence of some of these amino acids has also been considered essential in the development of antioxidant activity and this activity has become very important in recent years (HM Chen, K. Muramoto, F. Yamauchi, K. Fujimoto and K. Nokihara, Antioxidative properties of histidine-containing peptides designed from peptide fragments found in the digests of a soybean protein, Journal of Agricultural and Food Chemistry, 1998, 46: 49-53). Different degenerative diseases, such as cancer, Alzheimer's, cataracts, or aging itself are related to the oxidation of cellular components, lipids, proteins or DNA. These pathologies can occur as a result of the imbalance between oxidizing agents and the body's antioxidant systems, so the ingestion of antioxidant compounds in the diet could be useful in preventing such diseases. In addition, these antioxidant compounds present in food retard the processes of fat oxidation, which are considered responsible for alterations and the appearance of unpleasant odors and flavors in said foods. Recent research has revealed the ability of different milk proteins and peptides derived from them to exert antioxidant activity through various mechanisms of action. Thus, peptides with free radical chelating capacity from casein hydrolysates have been described (K. Suetsuna, H. Ukeda and H. Ochi, Isolation and characterization of free radical scavenging activities peptides derived from casein, Journal of Nutritional Biochemistry, 2000, 11: 128-131; EP1188767, Isolated antioxidant peptides from casein and methods for preparing, isolating and identifying antioxidant peptides) and whey proteins (B. Hernández-Ledesma, A. Dávalos, B. Bartolomé and L. Amigo, Preparation of antioxidant enzymatic hydrolyzates from α-lactalbumin and β-lactoglobulin Identification of active peptides by HPLC-MS / MS, Journal of Agricultural and Food Chemistry 2005, 53, 588-593). In addition, caseins have become an important source of peptides with inhibitory activity of the enzymes catalysing fat oxidation processes (SG Rival, S. Fornaroli, CG Boeriu and HJ Wichers, Caseins and casein hydrolysates. I. Lipoxygenase inhibitory properties, Journal of Agricultural and Food Chemistry, 2001, 49:
287-294).

Most of the studies carried out so far have focused on the biological activity of peptides derived from bovine caseins. However, there is little data on the biological activities exerted by peptides from caseins of other species, such as sheep or goats. JA Gómez-Ruiz, I. Recio, and A. Pihlanto (Antimicrobial activity of ovine casein hydrolysates. A preliminary study. Milchwissenschaft-Milk Science International 2005, 60: 41-45) described the potent dose-dependent inhibitory effect of activity metabolic of Escherichia coli JM103 exerted by β-casein hydrolysates. However, in this study the identification of the peptides responsible for this effect was not carried out. On the contrary, several sequences released from ovine caseins have been identified during the fermentation and maturation processes characteristic of Manchego cheese production and some of them have demonstrated IECA activity (JA Gómez-Ruiz, M. Ramos and I. Recio, Identification and formation of angiotensin-converting enzyme-inhibitory peptides in Manchego cheese by high-performance liquid chromatography-tandem mass spectrometry, Journal of Chromatography A, 2004, 1054: 269: 277). The IC 50 values (concentration that inhibits 50% of the enzyme activity) of these sequences were between 24.1 and 1275.4 µM. In this study the peptide with the highest ACE inhibitory activity corresponded to the? S2-casein f (205-208) fragment of the VRYL sequence (SEQ. ID. No. 11), which presented an IC 50 value of 24.1 µM (JA Gómez-Ruiz, M. Ramos, and I. Recio, Angiotensin converting enzyme-inhibitory activity of peptides isolated from Manchego cheese. Stability under simulated gastrointestinal digestion. Int. Dairy Journal 2004, 1075-1080). However, there is no data on the ability of these peptides to cross the intestinal barrier and on their ability to exert the antihypertensive effect in vivo . It should be noted that frequently, many peptides that are shown as potent ACE inhibitors in vitro lose all or part of their activity when they are tested in vivo , or even, peptides that in vitro do not exhibit great activity as ACEIs acquire it in vivo due to the performance of digestive enzymes (M. Maeno, N. Yamamoto and T. Takano, Identification of an anti-hypertensive peptide from casein hydrolysate produced by a proteinase from Lactobacillus helveticus CP790, Journal of Dairy Science, 1996, 79: 1316- 1321). There are also no studies on the multifunctional capacity of peptides released from caseins of different species to perform various biological activities, such as antihypertensive, antimicrobial and / or
antioxidant

Given the high biological quality of proteins of milk, it is of great interest to obtain, from these, bioactive peptides that consumed as part of the diet, in addition to exercise their basic nutritional functions, be able to produce metabolic or physiological effects, useful in the health maintenance and disease prevention. The production of bioactive peptides from the proteins in the milk would allow us to find new uses for this food, beyond its classic nutritional value, including the production of products Medicinal and nutraceutical. This would contribute to the development of healthy, safe and high quality foods, contributing to use and revaluation of dairy products.

Description of the invention Brief Description of the Invention

The present invention consists in the production of products derived from milk proteins that contain bioactive peptides with antimicrobial activity and / or ACEI activity in vitro and / or antihypertensive activity and / or antioxidant activity, by enzymatic hydrolysis of the casein fraction.

Bioactive peptides are produced by hydrolysis of one or more proteins, peptides or fragments of the themselves, which contain the amino acid sequence of said bioactive peptides (preferably containing α_ {s2} -casein), using enzymes preferably pepsin) and hydrolysis conditions that allow the breakdown of the protein chain in the right places for its release. They can also be obtained by chemical synthesis or by recombinant methods etc. Such peptides can be consumed as such, or from raw hydrolysates, of low molecular weight concentrates, or other subfractions assets obtained by size separation methods or chromatographic methods

These hydrolysates, their fractions or peptides, could be part of food products, acting as preservatives thereof, and reinforcing after ingestion the natural defenses of the organism. In addition, they could be used in development of pharmaceutical products, mainly intended for prevent and treat diseases, such as high blood pressure and / or bacterial infections. The invention extends the applications of milk proteins contributing to their use and revaluation.

Detailed description of the invention

The invention provides a method for producing bioactive peptides from milk caseins. Said bioactive peptides are those identified with the amino acid sequences shown in the SEQ. ID. . 1, SEQ. ID. . 2, SEQ. ID. . 3, SEQ. ID. . 4, SEQ. ID. . 5, SEQ. ID. . 6, SEQ. ID. . 7, SEQ. ID. . 8, SEQ. ID. No. 9 and SEQ. ID. No. 10 (table 1), some of which have antimicrobial activity and / or ACEI activity in vitro and / or antihypertensive activity and / or antioxidant activity.

The starting material of the present invention it would be any appropriate substrate that comprised one or more proteins or peptides, of animal, vegetable, or originating from microorganisms, which contain the amino acid sequence of Bioactive peptides of interest (SEQ. ID. No. 1, SEQ. ID. No. 2, SEQ. ID. . 3, SEQ. ID. . 4, SEQ. ID. . 5, SEQ. ID. . 6, SEQ. ID. . 7, SEQ. ID. . 8, SEQ. ID. No. 9 and SEQ. ID. No. 10, table 1), preferably α s2-ovine casein. Dice that all of them belong to the sequence of the α_ {s2} -casein, it is obvious that it could any preparation containing α_ {s2} -casein of different species, or α2s-casein peptides or fragments of any size, alone or mixed with other proteins. By example: α_ {s2} - pure casein, casein whole, caseinates and milk in its different forms of presentation, fermented dairy products, hydrolyzed milk proteins, dairy by-products, dairy products for animal feed, etc.

Said starting material dissolves or dispersed, at an appropriate concentration, in water or in a buffer solution, at a pH suitable for the enzyme's performance proteolytic Any proteolytic enzyme capable of break the protein present in the starting material and provide the peptides of interest, but preferably pepsin at pH 2.0-3.0. They could also be used proteolytic microorganisms that carried out a fermentation of the substrate and the hydrolysis of the protein.

Hydrolysis conditions: pH, temperature, enzyme-substrate ratio, disruption of reaction etc., are optimized in order to select the more active hydrolysates. In a particular embodiment, it obtain the bioactive peptides using pepsin at pH 3.0, in a enzyme / substrate ratio 3.7 / 100 (w / w) and performing hydrolysis at 37 ° C, for a period of time between 10 minutes and 24  hours, but preferably for a time less than 30 minutes

Then, if it is desired to concentrate the bioactive peptides, and since the peptides with antimicrobial activity possess cationic character, the separation of the fractions containing the bioactive peptides can be carried out by cation exchange chromatography (FPLC). From the higher cationic fractions, active subfractions can be isolated by a new step of cation exchange chromatography, hydrophobic chromatography, etc., or preferably high efficiency reverse phase liquid chromatography (RP-HPLC). Alternatively, from the hydrolyzate, the bioactive peptides can be concentrated by methods such as ultrafiltration, dialysis, electrodialysis with suitable pore membranes, gel filtration chromatography,
etc.

In addition to the complete hydrolysates and their fractions, the peptides shown in Table 1 and indicated with the SEQ. ID. . 1, SEQ. ID. . 2, SEQ. ID. . 3, SEQ. ID. . 4, SEQ. ID. . 5, SEQ. ID. . 6, SEQ. ID. . 7, SEQ. ID. . 8, SEQ. ID. No. 9 and SEQ. ID. No. 10, possess bioactive properties, fundamentally antimicrobial activity and / or ACEI activity and / or antihypertensive activity and / or antioxidant activity and are also object of the present invention. Specifically, the peptides identified with the SEQ sequences. ID. . 1, SEQ. ID. . 2, SEQ. ID. . 3, SEQ. ID. . 4, SEQ. ID. . 5, SEQ. ID. . 6, SEQ. ID. . 7, SEQ. ID. . 8, SEQ. ID. . 9 and SEQ. ID. . 10 have antimicrobial activity against Gram-positive bacteria and at least the SEQ sequence. ID. Nº 3 also exerts a potent antimicrobial effect against Escherichia coli . In addition, the peptides identified with the SEQ sequences. ID. . 1 and SEQ. ID. . 7 show a potent ACEI activity in vitro and the SEQ sequence. ID. . 7 has antihypertensive activity in spontaneously hypertensive rats (SHR) when administered orally to these animals. On the other hand, at least the peptide identified as SEQ. ID. . 7, has a remarkable antioxidant activity through a mechanism of chelation of oxygen radicals. It should be noted that these are natural peptides from products of wide consumption from which few side effects and good tolerance can be expected.

Likewise, the bioactive peptides identified in hydrolysates (SEQ. ID. No. 1, SEQ. ID. No. 2, SEQ. ID. No.. 3, SEQ. ID. . 4, SEQ. ID. . 5, SEQ. ID. . 6, SEQ. ID. . 7, I KNOW THAT. ID. . 8, SEQ. ID. No. 9 and SEQ. ID. No. 10, table 1) to know your sequence, the available technology allows you to Obtained by chemical and / or enzymatic synthesis of peptides or by recombinant methods

TABLE 1 Sequences of bioactive peptides identified

one

The obtaining of bioactive peptides from ovine α2S-casein hydrolysates with pepsin had not been previously described although antimicrobial peptides derived from this protein of bovine origin had been identified (EP 1114060, Process for producing cationic peptides from biological fluids). Some peptides derived from? S2-casein and other ovine caseins in Manchego cheese had also been previously identified with ACEI activity (JA Gómez-Ruiz, M. Ramos and I. Recio, Identification and formation of angiotensin-converting enzyme-inhibitory peptides in Manchego cheese by high-performance liquid chromatography-tandem mass spectrometry, Journal of Chromatography A, 2004, 1054: 269: 277), although its antihypertensive activity had not been studied in vivo . Among the peptides with ACEI activity previously identified is fragment 205-208 of the α-s2-ovine casein of the VRYL sequence (SEQ. ID. No. 11) (IC 50 24.1 µM). However, the sequence SEQ. ID. . 7 of the present invention, PYVRYL (IC 50 1.94) has an IECA activity 12 times more potent than previously described, which justifies the need for the entire sequence found in the present invention to exert a remarkable IECA activity. . In addition, the entire SEQ is also required. ID. . 7 to exert antihypertensive and / or antioxidant and / or antimicrobial activity.

These milk products: complete hydrolysates, the fractions thereof, or one or more of its constituent bioactive peptides (including its derivatives, their pharmaceutically acceptable salts and mixtures thereof), could be used as therapeutic substances with activity antimicrobial and / or ACEI activity and / or activity antihypertensive and / or antioxidant activity. Such products Dairy products can be subjected to heat treatment, such as pasteurization, or undergo drying or lyophilization etc., to be used as functional food products, additives or food ingredients, or pharmaceuticals, for the treatment and / or prevention of infections and / or hypertension arterial in all its forms, mainly in humans, although also in animals. The amount of hydrolyzate, fraction of low molecular weight, peptides, their derivatives or salts pharmaceutically acceptable and their mixtures, as well as their dosage for the treatment of some pathology, it will vary depending on numerous factors, such as age, severity of the pathology or dysfunction, route of administration and dose frequency. These Compounds could occur in any form of administration,  solid or liquid, and administered by any appropriate route, oral, respiratory, rectal or topical, although particularly designed for solid or liquid administration via oral.

In general, the process of obtaining these products: the complete hydrolysates, the fractions thereof and its constituent peptides, can be optimized, directing it to the production of as many bioactive peptides as possible or to control as much as possible the appearance of bitterness, originated normally by a high concentration of hydrophobic peptides of intermediate or low molecular weight.

Analytical procedures Measurement of antimicrobial activity

The antimicrobial activity is determined according to the method of A. Pellegrini, C. Deltting, U. Thomas, P. Hunziker (Isolation and characterization of four bactericidal domains in the bovine \ beta-lactoglobulin. Biochimica et Biophysica Acta, 2001, 1526 : 131-140), using as microorganisms Escherichia coli [American Type Culture Collection (ATCC), Rockville, MD, USA] ATCC 25922 , Listeria innocua [Spanish Type Culture Collection (CECT) Valencia, Spain] CECT 910T, Sthaphylococcus epidermidis CECT 231, Enterococcus faecalis CECT 795, Serratia marcescens CECT 854 and Sthaphylococcus carnosus CECT 4491T.

Bacterial suspensions are inoculated at 1% in the culture medium Triptosa-Soy (TSB) for Escherichia coli , Serratia marcescens and strains of the genus Sthaphylococcus , or in the brain-heart infusion broth (BHI) for Enterococcus faecalis and Listeria innocua . Incubation is carried out at 37 ° C, except in the case of Serratia marcescens , which is carried out at 30 ° C.

The bacterial inoculum, from which the work begins, is obtained after incubating a colony grown in TSB-Agar or BHI-Agar in 10 mL of TSB or BHI overnight at 37 ° C or 30 ° C. The bacterial suspension (1 mL) is diluted 1/50 with the corresponding culture medium, incubating at the appropriate temperature for each strain until reaching a population density of 1-4x108 colony forming units (CFU) per mL . The culture is centrifuged at 2000 x g for 10 minutes, the sedimented bacteria are washed twice with 15 mL phosphate buffer (pH 7.4) and the population is adjusted to 10 6 CFU / mL. In a sterile multiwell plate (Greiner Labortechnik, Frickenhausen, Germany) 50 µL of the bacterial suspension, 50 µL of the substance to be tested and 100 µL of the phosphate buffer are mixed with 2% of the appropriate culture medium in each case , and the mixture is incubated at 37 ° C or 30 ° C for 2 hours. After this time, the mixture is diluted to 10-5, 100 µL of each of the dilutions are added to TSB-Agar or BHI-Agar plates and the plates are incubated for 24 hours, after which time carries out the counting of
colonies

To calculate the antimicrobial activity, use the following formula:

Activity\ antimicrobial = log \ \ frac {N_ {0}} {N_ {f}},

where

N_ {0} corresponds to the number of CFU / mL initial

N_ {f} corresponds to the number of CFU / mL final

Measurement of the inhibitory activity of the converting enzyme of angiotensin (ACEI)

ACEI activity is measured in vitro according to the method of DW Cushman and HS Cheung (Spectrophotometric assay and properties of the angiotensin-converting enzyme of rabbit lung, Biochemical Pharmacology, 1971, 20: 1637-1648), subsequently modified by YK Kim , S. Yoon, DY Yu., B. Lónnerdal and BH Chung (Novel angiotensin-I-converting enzyme inhibitory peptides derived from recombinant human \ s_ {s2} -casein expressed in Escherichia coli . Journal of Dairy Research 1999, 66, 431 -439).

The substrate, hippyl histidyl leucine (HHL, Sigma, Chemicals Co, St. Louis, MO, USA), dissolves in buffer 0.1 M borate with 0.3 M NaCl, pH 8.3, to obtain a concentration 5 mM final. To 100 µL of substrate is added 40 µL of each one of the samples whose IECA activity is to be determined. Be add the enzyme ECA (EC 3.4.15.1, Sigma), dissolved in glycerol at 50%, and diluted at the time of testing 1/10 in water double distilled The reaction is carried out at 37 ° C, for 30 minutes in a water bath. The enzyme is inactivated by lowering the pH with 150 µL of 1N HCl. The formed hippuric acid is extracted with 1000 µL of ethyl acetate. After vortexing for 20 seconds, centrifuge at 3000 x g for 10 minutes and at room temperature. 750 µL of the organic phase is taken which is evaporate by heating at 95 ° C for 10 minutes. The residue of Hippuric acid is redissolved in 800 µL of double distilled water and, After stirring for 20 seconds, the absorbance at 228 nm is measured at a Dur-70 spectrophotometer from Beckman Instruments, Inc., Fullerton, USA.

To calculate the percentage of IECA activity, use the following formula:

% \ IECA = \ frac {Acontrol-Amuestra} {Acontrol-Ablanco} * 100

White is used to correct the background absorbance. This contains substrate, enzyme and 20 µl of double distilled water instead of sample, and the reaction is stopped in time  zero. The control supposes one hundred percent of the enzymatic action on the substrate in the absence of inhibitors, and contains 20 µl of water instead of sample and incubate the same time as the sample.

The results are presented as IC 50 (µM) or concentration at which 50% of activity is inhibited of the enzyme. The protein concentration is determined by the bicinconinic acid assay (Pierce, Rockford, IL, USA) using as bovine serum albumin pattern.

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Measurement of antioxidant activity

The oxygen radical absorption capacity (ORAC) is determined following the method developed by BX Ou, M. Hampsch-Woodill, RL. Prior (Development and validation of an improved oxygen radical absorbance capacity assay using fluorescein as the fluorescent probe, 2001, 49: 4619-4626). This method is based on the oxidation of fluorescein by peroxyl radicals produced in situ by thermal decomposition of 2,2'-azobis 2-amidinopropane dihydrochloride (AAPH). Oxidation of fluorescein causes a decrease in fluorescence at λ exc = 493 nm and λ em = 515 nm. The presence of antioxidants prevents or delays the breakdown of fluorescence.
Ceine

The working solution of fluorescein is prepares daily at a concentration of 60 nM from a 100 µM fluorescein stock solution in 75 mM phosphate buffer (pH 7.5). Acid is used as a reference antioxidant 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic (Trolox), which is prepared at a concentration of 20 mM (stock solution) in phosphate buffer and stored at -20 ° C. A curve of Trolox calibration by the analysis of the standard solutions of concentrations 12.5; 25; 40; 50 and 100 µM, prepared from of the stock solution. AAPH dissolves in phosphate buffer up to a final concentration of 143 mM, keeping low temperature to prevent its degradation.

To carry out the test, 375 are mixed µL of the sample with 375 µL of the AAPH and 2,225 mL of fluorescein, incubating this mixture at 37 ° C. Every 5 minutes is measured fluorescence (λ exc = 493 nm and λ em) = 515 nm) in the fluorimeter RF-1501 (Shimadzu). Be perform test controls consisting of a blank containing  fluorescein and phosphate buffer, to check the stability of the fluorescein during the experiment, and a positive control of maximum oxidation containing fluorescein, AAPH and phosphate buffer. As a control of the measure of antioxidant activity, a 40 µM trolox solution in each set of samples to be analyzed. All samples were analyzed in triplicate.

The antioxidant activity is quantified through of the measurement of the "area under the curve" (AUC) of the curve of fluorescein fluorescence decay and is expressed as Trolox equivalents (ORAC value). The AUC is calculated using The following formula:

AUC = (0.5 + f_ {5} / f_ {0} + f_ {10} / f_ {0} + f_ {15} / f_ {0} + ..... + f_ {30} / f_ {0})

Where f_ {0} is zero time fluorescence and f_ is the fluorescence at time i.

The relative ORAC value for the peptides is Determine with the following formula:

ORAC = [(AUC_ {sample} - AUC_ {white}) / (AUC_ {trolox} - AUC_ {white})] \ x \ [(\ trolox \ molarity / \ la \ molarity sample)]

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Isolation of peptide fractions by chromatography of ion exchange (FPLC)

Isolation of peptide fractions is carried carried out following the method of I. Recio, S. Visser (Identification of two distinct antibacterial domains within the sequence of bovine α_ {s2} -casein. Biochimica et Biophysica Act 1999, 1428: 314-326) with some modifications, in an FPLC equipment, using a column of cation exchange HiLoad? 26/10 SP Sepharose Fast Flow (Pharmacia, Uppsala, Sweden). Phases A and B consist of 10 mM NH 4 HCO 3 (adjusted to pH 7.0 with HCOOH) and NH 3 1.5 M, respectively. The samples dissolve in phase A prepared at a concentration of 5 mg / mL, injecting a volume 5 mL by a 50 mL Superloop ™ (Pharmacia). He Hydrolyzate elutes at a flow of 5 mL / min. After 20 minutes with 100% of solvent A, a gradient of 0 to 50% of solvent B is applied in A in 60 minutes, followed by 20 minutes with 50% of solvent B. The detection is carried out at an absorbance of 214 nm. The temperature of the column and the mobile phases is 9 ° C. The Fractions are collected after several chromatographic analyzes.

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Isolation of peptide fractions by chromatography High efficiency liquid in reverse phase (RP-HPLC) on a semi-preparatory scale

A team consisting of two pumps is used programmable model Waters Delta 600, a diode detector aligned model 966, a model 717 plus automatic injector, and a automatic fraction collector (Waters Corp., Mildford, MA, USA). A C 18 Prep NovaPack® HR column, 7.8 x 300 mm is used and 6 µm pore size (Waters), with a C_ {18} cartridge (Waters) as a column guard. The analyzes are performed at 30 ° C and the detection at 214 and 280 nm. The acquisition of the data takes out with the Millennium Software version 3.2 (Waters). The samples they are prepared at a concentration of 2.5 mg / mL and, prior to injection, centrifuged at 16,000 x g for 10 minutes. For the elution of the samples a binary gradient of water milliQ® (phase A) and acetonitrile (phase B) with 0.1% and 0.08% of trifluoroacetic acid, respectively, and a flow of 4 mL / min. He Phase B gradient is 0% to 40% in 50 minutes and 40% to 70% for 5 min, washing the column with 70% B for 5 minutes and reconditioning the column in the initial conditions for 25 min. The injected sample volume is 300 \ mul.

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Tandem mass spectrometry analysis (off-line)

Esquire 3000 ion trap equipment is used (Bruker Daltonik GmbH, Bremen, Germany). Samples are prepared at a concentration of 2 mg / mL in a solution of water: 50% acetonitrile (v / v) with 0.01% formic acid (v / v). The sample is injected into the electrospray nebulizer at a flow of 4 µl / min, using a syringe pump type 22 (Harvard Apparatus, South Natick, MA, USA). The equipment uses nitrogen as a nebulizer and drying gas, and operates with a helium pressure of 5 x 10-3 bar. Mass spectra are acquired in a range of 100-2000 m / z , and at a speed of 13000 Da / second. Tandem mass spectra are interpreted for the identification of peptide sequences using the Biotools 2.1 program (Bruker Daltonik GmbH, Bremen, Germany).

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Analysis using coupled RP-HPLC online to mass spectrometry in tandem (RP-HPLC-MS / MS)

Esquire-LC equipment (Bruker Daltonik GMBH, Bremen, Germany) is used. The HPLC equipment (1100 series) consists of a quaternary pump, an automatic injector, an eluent degassing system and a variable wavelength ultraviolet detector (Agilent Technologies, Waldbronn, Germany) coupled in line to a mass spectrometer of ionic trap Esquire 3000 (Bruker Daltonik). The column is a Hi-Pore C18 column (250 x 4.6 mm di, 5 µm particle size) (Bio-Rad Laboratories, Richmond, CA, USA). Solvent A is a mixture of water and trifluoroacetic acid (1000: 0.37) and solvent B a mixture of acetonitrile and trifluoroacetic acid (1000: 0.27). 50 µL of prepared sample is injected at a concentration of 4.5 mg / ml. A flow of 0.8 ml / min is used, with a linear gradient from 0% to 50% of solvent B in A in 60 minutes. The eluent is monitored at 214 nm, by mass spectrophotometry, under the same conditions as indicated in the previous section, except that the injection flow of the sample through the nebulizer is of
275 µl / min.

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Study of antihypertensive activity in rats spontaneously hypertensive (SHR)

The effect of several of the peptides is studied identified on the blood pressure of SHR rats. Peptides They are chemically synthesized for this study.

The study is carried out with male SHR rats, of 17-20 weeks of age, and weight between 300 and 350 g, from Charles River Laboratories España S.A. The rats are stored in cages five by five, and kept at a stable temperature of 25ºC, with cycles of 12-hour light-dark, ingesting water and food freely available Blood pressure measurements are carried out systolic (PAS) and diastolic (PAD), and for this the tail cuff method (RD. Buñag, Validation in awake rats of a tail-cuff method for measuring systolic pressure, J. Appl. Physiol., 1973, 34: 279-282). The equipment used (Le5001, Letica) provides digital values of the PAS and the PAD automatically. This equipment also records and facilitates the frequency measurement Animal cardiac Before placing the sleeve and the transducer in the tail of rats, these are exposed to a temperature close to 37 ° C to facilitate dilation of the caudal artery. In addition, to ensure the reliability of the measure, the animals get used to the procedure 2 weeks before Carry out the test in question. The value of the PAS and the PAD It is established by performing 3 consecutive measurements, and calculating the average of the three values corresponding to each of these two variables

The SHR rats used for the study had PAS values between 190 mm Hg and 220 mm Hg, and values of PAD between 130 mm Hg and 180 mm Hg.

The administration of the products to be tested is carried out by means of an intragastric tube in a period of time between 9 and 10 am, and the dose tested is administered dissolved in 1 ml of distilled water. PAS and PAD measurements are taken before administration, and periodic measurements of these variables are also taken every 2 hours after administration, up to 8 hours post-administration. Additionally, PAS and PAD measures are also taken 24 hours after the administration of said products. As a negative control (to establish the circadian variation of the PAS and the PAD in probed rats) the measures of the PAS and the PAD obtained in similar tests with rats are used, to which 1 ml of water is administered by intragastric tube. As a positive control, the PAS and PAD measurements obtained in similar trials with rats that are administered by intragastric tube 50 mg / kg of captopril (prototype IECA drug) are used. This dose of captopril is administered to each rat dissolved in 1 ml of water.
distilled

The results are grouped and the average is obtained ± the standard error of the mean (ESM) for a minimum of 6 homogeneous tests. To compare them, an analysis of one-way variance, followed by the Bonferroni test. Is considered significant difference for values of p <0.05.

Brief description of the content of the figures

Figure 1 represents a chromatogram, obtained using cation exchange chromatography (FPLC), of α2 s-ovine casein hydrolyzate with pepsin for 30 minutes, in which 5 fractions are selected (FA-FE), which were collected manually. At Abcissa axis represents the time in minutes.

Figure 2A is a chromatogram, obtained using high performance liquid chromatography in phase inverse (RP-HPLC) at semi-preparatory scale, of the FC fraction collected from the hydrolyzate of α_ {s2} -ovine casein with pepsin for 30 minutes 4 subfractions are selected (FC1-FC4), They were collected automatically. On the axis of abscissa is Represents the time in minutes.

Figure 2B is a chromatogram, obtained using high performance liquid chromatography in phase inverse (RP-HPLC) at semi-preparatory scale, of the fraction FD collected from the hydrolyzate of α_ {s2} -ovine casein with pepsin for 30 minutes 2 subfractions are selected (FD1-FD2), They were collected automatically. On the axis of abscissa is Represents the time in minutes.

Figure 3 represents the activity antimicrobial of the different subfractions obtained from FC and FD fractions using RP-HPLC at scale semi-preparative

Figure 4 represents the decrease in systolic blood pressure (SBP), and the decrease in diastolic blood pressure (PAD), obtained in spontaneously hypertensive rats, after administration by intragastric tube of 1 ml of water (?), 50 mg / kg of Captopril (\ Box), 3 mg / kg of PYVRYL (\ triangle), and 3 mg / kg of LKKISQ (\ blacklozenge). T (h) represents the time elapsed since the administration expressed in hours. Data represent the mean ± ESM for a minimum of 6 animals. a P <0.05 vs water; b P <0.05 vs Captopril; c P <0.05 vs PYVRYL.

Examples of embodiment of the invention

The following examples illustrate the invention, although they should not be considered as limiting the scope of the same.

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Example one

Obtaining bioactive peptides, with antimicrobial activity, ACEI, antihypertensive and antioxidant, from the hydrolyzate of α2 sine sheep casein with pepsin

The hydrolyzate was obtained using α-s2-ovine casein as a substrate, obtained after separation of the rest of the caseins by the method of HJ Vreeman, JAM van Riel (The large-scale isolation of α_ {s2} -casein from Bovine Casein, Netherlands Milk and Dairy Journal, 1990, 44: 43-48). As an enzyme, porcine pepsin (EC 3.4.23.1. 570 U / mg protein), from pork stomach (Sigma Chemical, St. Louis, USA) was used. An aqueous solution of 0.5% sheep α-s2-casein was prepared and the pH adjusted to 3.0 with 1 M HCl. Pepsin (enzyme / substrate ratio 3.7 / 100, p / p). Hydrolysis was carried out at 37 ° C for 30 minutes. Pepsin inactivation was achieved by heating at 80 ° C for 15 minutes and subsequently adjusting the pH to 7.0 with 1 M NaOH. The supernatant collected after centrifugation of the hydrolyzate at 16000 g for 15 minutes at 5 ° C was analyzed by FPLC (Figure 1), five fractions (FA-FE) were separated, which were collected manually and subsequently
lyophilized

The antimicrobial activity of these five fractions was measured at a concentration of 2.5 mg / mL, using as a control E. coli at 5.9 × 10 5 CFU / mL. The results revealed that the FC and FD fractions had antimicrobial activity, reducing the number of microorganisms by 2.54 and 0.6 orders of magnitude, respectively.
mind.

In order to identify the peptides responsible for antimicrobial activity, the FC and FD fractions were analyzed by RP-HPLC on a semi-preparative scale. The chromatographic profile of the FC fraction (Figure 2A) and the FD fraction (Figure 2B) are shown in Figure 2. Four subfractions (FC1-FC4) were separated from the FC fraction and two subfractions (FD1-FD2) from the FD fraction. Each of these subfractions were collected and, after evaporation of acetonitrile, they were lyophilized. The antimicrobial activity of these subfractions was measured at a concentration of 2.5 mg / mL against E. coli (6.2 x 10 6 CFU / mL). Figure 3 shows the values of antimicrobial activity against E. coli of these subfractions. Of all the subfractions, it is worth highlighting FC1, which was the one that exhibited the highest antimicrobial activity, since it had a bactericidal effect at the concentration tested (log N f / N 0 greater than 6). Subfractions FC4, FD1 and FD2 showed a moderate antimicrobial activity, with reduction values of the number of microorganisms of 1.24, 1.31 and 1.64 orders of magnitude,
respectively.

Subfractions FC1, FC4, FD1 and FD2 are analyzed by mass spectrometry, using an analyzer of ionic trap following the previously described methodology. The Identified peptides are shown in Table 1.

TABLE 1 Peptides identified in subfractions FC1, FC4, FD 1 and FD2 obtained from the hydrolyzate of α_ {s2} -ovine casein with pepsin for 30 minutes

2

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Example 2

Peptides obtained by chemical synthesis with activity antimicrobial

The peptides were chemically synthesized majority present in the subfractions obtained from α2 s-ovine casein hydrolyzate with Pepsin for 30 minutes (SEQ. ID. No. 1; SEQ. ID. No. 2; SEQ. ID. . 3 and SEQ. ID. No. 7). These peptides were synthesized by the solid phase Fmoc method and its purity was verified by RP-HPLC-MS / MS.

The antimicrobial activity of synthetic peptides was measured at a concentration of 0.5 mM, against Escherichia coli , Serratia marcescens , Staphylococcus carnosus, Staphylococcus epidermidis, Enterococcus faecalis and Listeria innocua . The activity results are shown in table 2.

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TABLE 2 Antimicrobial activity of synthetic peptides identified in subsections FC1, FC4 and FD1 obtained at from the α-s2-casein hydrolyzate sheep with pepsin for 30 minutes

3

The antimicrobial activity of these peptides against Gram positive bacteria is high, especially against the tested species of the genus Staphylococcus . Three of these SEQ peptides. ID. . one; I KNOW THAT. ID. Nº 2 and SEQ. ID. . 3 presented bactericidal activity against S. carnosus . However, Gram negative bacteria ( E. coli and S. marcescens ) are very resistant to the action of all these peptides, although it should be noted that the peptide identified as SEQ. ID. . 3 showed a high antimicrobial activity against E. coli .

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Example 3

Peptides obtained by chemical synthesis with ACEI activity and antihypertensive

The ACEI activity of two of the chemically synthesized peptides, specifically the sequences I KNOW THAT. ID. . 1 and SEQ. ID. No. 7, mentioned in example 1. The activity results, expressed as IC 50, or concentration protein needed to inhibit 50% of enzyme activity They are shown in Table 3.

The two peptides have a potent activity IECA

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TABLE 3 ACEI activity of synthetic peptides identified in subsections FC 1 and FD 1 obtained at from the α-s2-casein hydrolyzate sheep with pepsin for 30 minutes

4

The antihypertensive activity of the SEQ peptides. ID. . 1 and SEQ. ID. . 7, for which it administered said peptides (3 mg / kg) to SHR rats. Peptides they were dissolved in distilled water and the corresponding dose was administered to each rat in a volume of 1 mL.

Figure 4 shows the decreases in SBP. and of the PAD obtained in SHR rats, at different times, after 3 mg / kg administration of SEQ peptides. ID. . 1 and SEQ. ID. . 7. It can be seen that administration of the SEQ ID peptide Nº 7 causes a significant decrease in the PAS and the PAD in These animals. The decrease of these variables is maximum 4 hours after administration of this peptide The decrease it also presents a temporary course similar to that of the decrease of PAS and PAD produced by the administration of captopril, which is a compound of proven antihypertensive activity. These results show that the peptide identified by the sequence I KNOW THAT. ID. . 7 has a clear and pronounced antihypertensive effect when administered acutely orally.

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Example 4

Peptides obtained by chemical synthesis with activity antioxidant

The antioxidant activity of the sequence SEQ. ID. . 7, mentioned in example 1. The value of chelating activity of peroxyl radicals is shown at continuation:

ORAC_ {PYVRYL} = 1 82 \ \ mol mol \ Trolox \ equivalents / \ mol mol \ peptide

The results show, therefore, that the PYVRYL peptide (SEQ. ID. No. 7) has an antioxidant activity 1.82 times higher than the activity of 1 µmol of Trolox.

<110> SUPERIOR INVESTIGATION COUNCIL SCIENTISTS

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Amigo Garrido, Lourdes

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Friend Garrido, Lourdes

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Ramos González, Mercedes

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Ramos González, Mercedes

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Aleixandre de Artiñano, Amaya

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<120> BIOACTIVE PEPTIDES IDENTIFIED IN ENZYMATIC HYDROLYZES OF DAIRY CASEINS AND PROCEDURE OF OBTAINING.

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Claims (21)

1. Bioactive peptide characterized by

to.

possess antimicrobial activity and / or ACEI activity in vitro and / or antihypertensive activity in vivo and / or antioxidant activity

b.

present in enzymatic hydrolysates of milk caseins with pepsin and

C.

the amino acid sequences of the following group: SEQ. ID. . 1, SEQ. ID. No. 2, SEQ. ID. . 3, SEQ. ID. . 4, SEQ. ID. . 5, SEQ. ID. . 6, SEQ. ID. . 7, SEQ. ID. . 8, SEQ. ID. No. 9 and SEQ. ID. No. 10.

2. Bioactive peptide according to claim 1 of SEQ sequences. ID. . 2, SEQ. ID. . 5, SEQ. ID. . 6, SEQ. ID. . 7, SEQ. ID. . 8, or SEQ. ID. No. 10 characterized by understanding the SEQ. ID. . 7. 3. Bioactive peptide according to claim 1 of SEQ sequences. ID. . 1, SEQ. ID. . 3, SEQ. ID. . 4, or SEQ. ID. No. 9 characterized by understanding the SEQ. ID. . one. 4. Bioactive peptide according to claims 1 to 3 characterized by presenting antimicrobial activity against Gram-positive bacteria. 5. Bioactive peptide according to claims 1 to 3 characterized by the amino acid sequence SEQ. ID. Nº 3 and present antimicrobial activity against Gram-negative bacteria such as Escherichia coli . 6. Bioactive peptide according to claims 1 to 3 characterized by the amino acid sequence SEQ. ID. Nº 1 or SEQ. ID. . 7 and present ACEI activity in vitro . 7. Bioactive peptide according to claims 1 and 2 characterized by the amino acid sequence SEQ ID No. 7 and for presenting antihypertensive activity. 8. Bioactive peptide according to claims 1 and 2 characterized by the amino acid sequence SEQ ID No. 7 and for presenting antioxidant activity by chelation of oxygen radicals. 9. Bioactive peptide according to claims 1 to 8, characterized in that it is obtained by a chemical or enzymatic synthesis process or by recombinant methods. 10. Bioactive peptide according to claims 9, characterized in that it is obtained by an enzymatic hydrolysis process of? S2-casein or whole casein or milk in its different forms of presentation, milk by-products, or fermented milk products. 11. Method for producing any of the bioactive peptides according to claim 1 to 8, characterized by:

to.

obtained by hydrolysis of the material Starting that would be any appropriate substrate containing one or more more proteins or peptides, of animal or vegetable origin, or from of microorganisms, preferably α2 s-casein or casein or whole milk, whose amino acid sequence comprised the sequence of amino acids of any of the bioactive peptides of interest indicated in claim 1,

b.

dissolve or disperse said material Starting at an appropriate concentration, in water or in a buffer solution, at a pH suitable for the enzyme's performance proteolytic,

C.

any proteolytic enzyme is used capable of breaking the protein present in the starting material and provide the peptides of interest, but preferably pepsin to pH 3.0; or proteolytic microorganisms that will carry out a substrate fermentation, and

d.

he reaction time would be between 10 minutes and 24 hours, but that preferably out of 30 minutes.

12. Bioactive peptide according to claims 1 to 3 characterized in that the methods indicated in claim 11 are used for obtaining it. 13. Bioactive product characterized in that it contains at least some of the peptides indicated in claims 1 to 3 as it is the enzyme hydrolyzate itself, any of its fractions, or a purification thereof.

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14. Bioactive peptide characterized by being pharmaceutically acceptable derivatives or salts, or mixtures of any of the bioactive products indicated in claims 1 to 10 and 12 and 13. 15. Pharmaceutical composition characterized by comprising at least some of the bioactive products with antimicrobial activity and / or ACEI activity in vitro and / or antihypertensive activity in vivo and / or antioxidant activity according to claims 1 to 10 and 12, 13 and 14. 16. Additive, ingredient, or functional food supplement characterized by comprising at least some of the bioactive products with antimicrobial activity, ACEI in vitro and / or antihypertensive activity in vivo and / or antioxidant activity according to claims 1 to 10 and 12, 13 and 14. 17. Functional food product characterized by comprising at least some of the bioactive products with antimicrobial activity, ACEI in vitro and / or antihypertensive activity in vivo and / or antioxidant activity according to claims 1 to 10 and 12, 13 14 and 16. 18. Pharmaceutical composition according to claim 15 for use in the manufacture of a medicament intended for the prevention and / or treatment of infections microbial 19. Pharmaceutical composition according to claim 15 for use in the manufacture of a medicament intended for the treatment of hypertension. 20. Use of additive, ingredient, food functional according to claim 16 in the preparation of a favorable functional food product to prevent infections microbial 21. Use of additive, ingredient, food functional according to claim 16 in the preparation of a favorable functional food product to mitigate the hypertension.